Transmissible Spongiform Encephalopathies (TSEs) are caused by a group of related viruslike agents that cause neurodegeneration in sheep, humans, cows and wild animals. Some strains of these agents have developed increased virulence, as evidenced by their epidemic spread. This application continues to investigate differences among agent strains in our animal models, and in our newly developed tissue culture models. We will exploit tissue culture models to clarify agent-specific properties on a biological as well as a structural level. We have established models of human CJD agents, including the BSE-linked human vCJD strain propagated in mice as well as in mpnotypic cells in culture. We also have successfully passaged the UK BSE agent for comparison, in addition to two reference sheep scrapie agents. Additional human CJD isolates from Japan have also been propagated in mice and cultures. We have found new ways to define these individual agent strains through the use of rapid interference bioassays in vitro. These tests reveal a new dimension of agent-specific properties. We will use these assays to determine the relative virulence and protective capacities of different environmental TSE agents. Our broad aim is to uncover agent properties that have predictive value for understanding the spread of emergent and epidemic TSE agents in nature. We will also develop high throughput live cell assays for infectivity in order to advance systematic analyses of the infectious agent, including purification of 25nm viruslike particles from highly infectious cells. We suspect these structures are the causal virions in all TSEs, including those propagated from families with a greater susceptibility to infection. Many viruses interact with host molecules to provoke pathological changes, and we think this is the origin of host prion protein (PrP) amyloid. Because PrP amyloids show no significant infectivity, it is important to evaluate other concepts of the infectious agent. Rapid infectivity assays in vitro can open the study of TSEs by providing a way to test any pure molecule or structure for its intrinsic infectivity by Koch's established postulates for infectious pathogens.